Increased activation of NADPH oxidase 4 in the pulmonary vasculature in experimental diaphragmatic hernia

Persistent pulmonary hypertension remains a major cause of mortality and morbidity in congenital diaphragmatic hernia (CDH). NADPH oxidases (Nox) are the main source of superoxide production in vasculature. Nox4 is highly expressed in the smooth muscle and endothelial cells of the vascular wall and increased activity has been reported in the pulmonary vasculature of both experimental and human pulmonary hypertension. Peroxisome proliferator-activated receptor (PPAR gamma) is a key regulator of Nox4 expression. Targeted depletion of PPAR gamma results in pulmonary hypertension phenotype whereas activation of PPAR gamma attenuates pulmonary hypertension and reduces Nox4 production. The nitrofen-induced CDH model is an established model to study the pathogenesis of pulmonary hypertension in CDH. It has been previously reported that PPAR gamma-signaling is disrupted during late gestation and H2O2 production is increased in nitrofen-induced CDH. We designed this study to investigate the hypothesis that Nox4 expression and activation is increased and vascular PPAR gamma is decreased in nitrofen-induced CDH. Pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into control and CDH. RT-PCR, western blotting and confocal-immunofluorescence-double-staining were performed to determine pulmonary expression levels of PPAR gamma, Nox4 and Nox4-activation (p22(phox)). There was a marked increase in medial and adventitial thickness in pulmonary arteries of all sizes in CDH compared to controls. Pulmonary Nox4 levels were significantly increased whereas PPAR gamma levels were decreased in nitrofen-induced CDH compared to controls. Western blotting revealed increased pulmonary protein expression of the Nox4-activating subunit p22(phox) and decreased protein expression of PPAR gamma in CDH compared to controls. Confocal-microscopy confirmed markedly increased pulmonary expression of the Nox4 activating subunit p22(phox) accompanied by decreased perivascular PPAR gamma expression in lungs of nitrofen-exposed fetuses compared to controls. To our knowledge, the present study is the first to report increased Nox4 production in the pulmonary vasculature of nitrofen-induced CDH. Down-regulation of the PPAR gamma-signaling pathway may lead to increased superoxide production, resulting in pulmonary vascular dysfunction and contributing to pulmonary hypertension in the nitrofen-induced CDH model. PPAR gamma-activation inhibiting Nox4 production may therefore represent a potential therapeutic approach for the treatment of pulmonary hypertension in CDH.